Bacterial cytokinesis is mediated by the septal ring, a cytoskeletal-like organelle that is associated with the cytoplasmic membrane at the site of division. The long-term goals of the proposed research are to understand at a molecular level: i) The composition and architecture of the organelle, ii) How the ring assembles from its different components, iii) How the proper site for its assembly is determined, and iv) How the ring drives cell constriction. In E.coli, the organelle consists of at least nine essential division proteins that co-assemble in a specific order to form the mature structure. The first recognized step in the formation pathway is self-assembly of the tubulin-like GTPase FtsZ on the membrane. FtsZ polymers are bound by the ZipA and FtsA proteins, resulting in a tripartite intermediate that is required for recruitment of the other components. Septal ring assembly in the cell is controlled by MinC, an inhibitor of FtsZ polymerization. The activity of MinC, in turn, is controlled by the MinD and MinE proteins. The latter two cause MinC to rapidly oscillate from pole-to-pole, forcing FtsZ assembly to the middle of the cell. Evidence indicates that MinD, in addition to driving oscillation of MinC, also specifically targets the division inhibitor to early septal ring intermediates, which are misplaced and destined for destruction. The DicB protein of bacteriophage Kim is also a potent stimulator of MinC activity, and appears to target MinC to its substratein a similar manner. It is proposed to elucidate the mechanisms whereby MinD and DicB help to target MinC activity, and to characterize the composition of the target(s). For this and other purposes it is proposed to develop in vitro systems wherein the properties of early septal ring complexes may be studied in their 'natural' membrane-associated state. Evidence further indicates that, in addition to the known division factors, a number of components are missing from the current septal ring model. Some of these must also be early components and be responsible for the onset of septal murein synthesis. It is proposed to identify additional septal ring components by determining the cellular distribution of several good candidates.